Download Effect of Ethanol Root Extract of Equisetum arvense

Zhang et al
Tropical Journal of Pharmaceutical Research August 2015; 14 (8): 1451-1458
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
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Available online at http://www.tjpr.org
http://dx.doi.org/10.4314/tjpr.v14i8.18
Original Research Article
Effect of Ethanol Root Extract of Equisetum arvense (L) on
Urinary Bladder Activity in Rats and Analysis of Principal
Plant Constituents
Haiying Zhang1, Ning Li1, Kun Li2,3 and Peng Li1*
1
2
Department of Urology of Yantai Hill Hospital, No. 91 of Jiefang Road, Yantai City, 264001, Department of Gynecology and
Obstetrics of Affiliated Hospital of Shandong Academy of Medical Sciences, No. 38, Wuyingshan Road, Jinan City, 250031,
3
Shandong Academy of Medical Sciences, No. 89, Jingshi Road, Jinan City, Shandong Province, 250062, China
*For correspondence: Email: [email protected]
Received: 28 October 2014
Revised accepted: 11 June 2015
Abstract
Purpose: To investigate the mechanism of action by which ethanol root extract of Equisetum arvense
influences urinary bladder activity in rats, and to characterize the major compounds of the extract.
Methods: Ethanol (95 %) was used for hot extraction for 3 h in a Soxhlet extraction apparatus. A total of
36 female rats were divided into Equisetum arvense root extract-treated group (EA) and control group.
Rats in EA group were treated with a standard diet containing 0.2 % of the extract, while rats in the
control group were fed with the diet only. After 3 weeks, 12 rats underwent cystometry with 0.2 % acetic
acid solution and bladder activity was recorded. In another 12 rats, blood pressure, body weight and
adenosine triphosphate were measured. In the remaining 12 rats, 0.2 % acetic acid solution was infused
into the bladder and urinary adenosine triphosphate determined before and after the stimulation.
Results: The results showed that during cystometry with acetic acid, the time interval between urinary
bladder contractions was shorter and maximum bladder contraction pressure was much greater in rats
in the control group, but in the Equisetum arvense group, the changes were much lower. Also in the
Equisetum arvense group, plasma adrenaline and noradrenaline levels were lower than for the control
group. Furthermore, increase in the levels of urinary adenosine triphosphate was smaller in Equisetum
arvense group than in control group.
Conclusion: This study suggests that Equisetum arvense ethanol root extract influences urinary
bladder activity by decreasing adenosine triphosphate release, and is therefore a potential therapeutic
agent against bladder disorder. However, there is need for further investigation of its exact mechanism
of action.
Keywords: Equisetum arvense, Bladder activity, Cystometry, Adenosine triphosphate release,
adenosine triphosphate, Liquid chromatography–tandem mass spectrometry
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INTRODUCTION
In recent years, interest in problems related to
urinary bladder control such as overactive
bladder and urinary leakage is on a rapid rise.
This increased interest may be linked to the
increased awareness of the deleterious effect of
lower urinary tract symptoms (LUTS) on quality
of life as well as the World Health Organization
(WHO) sponsored activities related to such
issues [1]. Overactive bladder which now is
thought to be a chronic condition is defined
urodynamically as detrusor overactivity, and
characterized by involuntary bladder contractions
Trop J Pharm Res, August 2015; 14(8): 1451
Zhang et al
during the filling phase of the micturition cycle [2].
Further, bladder overactivity is also reported in
benign prostatic hyperplasia (BPH), which is a
common condition in old men characterized by
enlargement of the prostate gland and is
frequently accompanied by lower urinary tract
symptoms (LUTS) and urinary tract obstruction
[3]. An increase in sympathetic tone and high
blood pressure are reported to complicate LUTS.
Further, many published studies suggest that
ATP secreted by epithelial cells of the bladder
also affects bladder activity.
There are several reports on the efficacy and
safety of phytotherapeutic compounds for BPH
and LUTS. Saw Palmetto extract from Serenoa
repens is one of the frequently studied and used
herbal agents [4]. Further, herbal medicinal
agents and their metabolites such as β-sitosterol,
Pygeum (extract from Prunus africana), and
Urtica dioica root extract. Gosha-jinki-gan, which
is a Chinese herbal medicine has been used for
BPH and LUTS. In animal studies using
continuous cystometry, Gosha-jinki-gan was
reported to increase the interval between bladder
contractions and increase bladder capacity [5-8].
However, only a limited number of therapies are
available for these urological disorders coupled
with the drawbacks of some current therapeutic
agents and there is a need for such therapeutic
targets which will be effective with less toxicity.
Therefore, the objective of this study was to
evaluate the effect and possible mechanistic
mechanism of action of the ethanol extract of
Equisetum arvense on urinary bladder activity in
rats and to determine bioactive compounds in the
extract.
EXPERIMENTAL
Materials
A total of 36 female Sprague-Dawley rats
(Dashuo experiment animal center, Chengdu)
weighing 150 - 210 g were used in this study.
The rats were divided into two groups, with a
control group of 18 rats and an Equisetum
arvense (EA) group of 18 rats. Rats in the control
group were fed a standard diet, while animals in
the EA group were fed with the same standard
diet along with 0.2 % Equiseteum arvense
ethanol extract powder. The rats were fed the
respective diets for 3 weeks and tap water was
freely accessible to them during this period. The
animals were maintained in accordance with the
U.S. National Institutes of Health Guide for the
Care and Use of Laboratory Animals (NIH
Publication 80-23, revised 1996) and all
procedures were approved by the Ethical
Committee of the General Hospital of Chengdu
Military
Region
(approval
ref
no.
IECA/GHC/2013/097).
Plant material and preparation of the extract
The roots of E. arvense were collected during
May - June 2013 from Jiuzhaigou, Chengdu,
China. The plant material was identified by Prof
Long Qing-De of the Guiyang Medical University
Department of Medicine, and a voucher
specimen (no. GMU-0077654) was deposited at
the herbarium of Guiyang Medical University,
Guiyang, China. The roots were thoroughly
washed with tap water, shade dried and then
chopped into small pieces. Ethanol (95 %) was
used for hot extraction carried out for 3 h using a
Soxhlet extractor. The extract was then
concentrated under reduced pressure in a rotary
evaporator at 40 oC and was then kept in a
refrigerator at 4 oC prior to use.
Body weight measurement and continuous
cystometry
After 3 weeks of feeding, 12 rats, 6 in the control
group and 6 in the EA group, were anesthetized
with urethane (0.5 gm/kg and 0.3 gm/kg
subcutaneously
and
intraperitoneally
respectively) and the body weight was
measured. To perform continuous cystometry, a
PE-50 polyethylene catheter was injected into
the bladder through the urethra. Physiological
saline was infused through the catheter and
bladder activity was measured through the
urethral catheter, which was connected to a
pressure transducer and a saline infusion pump
via a 3-way stop cock. Cystometry was continued
for at least 50 min and the interval between
bladder
contractions,
maximum
bladder
contraction pressure, intravesical baseline
pressure and bladder contraction time were
monitored and measured during the final 30
minutes. These rats were also subjected to
continuous cystometry with 0.2 % acetic acid
solution after continuous cystometry with
physiological saline was complete. Here again,
continuous cystometry with 0.2 % acetic acid
solution was continued for 50 min and similar
measurements were performed.
Evaluation of biochemical profile and blood
pressure
In the other 12 rats including 6 each from the
control and EA groups, blood pressure
measurement and biochemical tests were carried
Trop J Pharm Res, August 2015; 14(8): 1452
Zhang et al
out. Blood pressure was measured in conscious
and restrained rats by the tail cuff method using
a BP-98A sphygmomanometer. The blood
pressure was measured as the mean of 3
recordings obtained at 1-min time periods.
After this, each rat was anesthetized with 1.5 %
halothane and blood was withdrawn. The blood
sample
was
centrifuged
followed
by
measurement of the noradrenaline, adrenaline,
dopamine and serotonin levels in blood plasma
using high performance liquid chromatography
(HPLC) in combination with tandem mass
spectrometry.
Adenosine triphosphate (ATP) measurement
From the remaining 12 rats, including 6 rats each
from control group and 6 rats from EA group,
urine samples were collected. After this, the rats
were anesthetized with 1.5 % halothane while 2
ml of 0.2 % acetic acid solution was infused into
the bladder through a catheter for 15 min to
provide stimulation. To prevent any urinary tract
infection, the rats were injected subcutaneously
with 100 mg cefazolin sodium hydrate.
Spontaneously voided urine was also collected at
3-6 h (day 0) after recovery from halothane
anesthesia as well as on days 2, 4 and 6 after
the bladder stimulation. Urinary ATP was
measured using an ATP Hygiene kit HS and
Gene Light 55.
Liquid chromatography–tandem mass
spectrometry (LC–ESI-MSMS)/HPLC analysis
LC–MS equipment (LC–MS QqQ-6410B Agilent
Technologies) consisted of a chromatographic
system (1260 Infinity Agilent Technologies)
coupled with an Agilent Triple Quad mass
spectrometer fitted with an ESI source. MS
conditions were the following: MS range 100–
1200 Da, MSn spectra were obtained using both
positive and negative modes, nebulizer gas 45
Psi, gas temperature 325 oC, capillary voltage
4000 V.
HPLC analysis was carried out by an Agilent
1260 infinity series. A Chromolith RP-18e column
(4.6 mm ID, 50 mm length) (Merck) was used.
Mobile phase consisted of (A) aqueous formic
acid (0.1 %) and (B) methanol. Gradient
condition was; 0–8 min, linear gradient from 12 to
25 % of B; 8 – 12 min, isocratic conditions at 25
% of B; 12 – 16 min, linear gradient from 25 to 40
% of B; 16–40 min, linear gradient from 40 to 50
% of B, 40 – 50 min, linear gradient from 50 to
100 % of B. Flow rate was 1 ml/min.
Statistical analysis
The results are reported as mean ± standard
deviation (SD). Slide write Plus version 7.01 was
used for analysis of the data while Student’s
unpaired t-test was applied for statistical analysis
with p < 0.05 considered to be statistically
significant.
RESULTS
Effect of EA on the body weight of rats
There was no remarkable difference between the
body weights of the control and EA group of rats
before the experiment was initiated. However,
after 3 weeks of feeding on EA diet, body weight
of the rats in the EA group was remarkably
increased in comparison to the control diet group
rats (Table 1) (10 % increase, p = 0.015)
Continuous cystometry
There was no considerable difference in the time
interval
between
bladder
contractions,
intravesical
baseline
pressure,
bladder
contraction time and residual urine volume
between the EA group and control group rats
during continuous cystometry with physiological
saline. However, it is important to mention that
maximum bladder contraction pressure was
considerably lower by 21.7 % in the control group
as compared to the EA group (p = 0.01, Table 1).
However, during the continuous cystometry with
0.2 % acetic acid solution, it was observed that
the time interval between urinary bladder
contractions became considerably shorter by
18.5 % (p = 0.02) and maximum bladder
contraction pressure was significantly higher by
7.4 % (p = 0.01) in the control group as
compared to the EA group (Fig 1).
Biochemical profile and blood pressure
It was also observed that there were no
considerable differences in the bladder weight
and systolic, mean and diastolic blood pressure
between the two groups (control group and EA
group). The biochemical tests revealed that there
were small differences in the plasma dopamine,
ATP and serotonin between the two groups. But
it was revealed that plasma adrenaline and
noradrenaline concentrations were significantly
higher by 38.7 and 41.2 % in the control group as
compared to the EA group (p < 0.01 and p <
0.05, respectively, Table 2).
Trop J Pharm Res, August 2015; 14(8): 1453
Zhang et al
Figure 1: Intravesical infusion of E. arvense extract resulted in increased voiding frequency (decreased
intercontraction interval). Representative continuous cystometrogram recordings in rats treated with intravesical
protamine sulfate in control rats (A) and different concentrations of the extract (B-D).
Table-1: Impact of Equisetum arvense ethanol extract on continuous cystometry with physiological saline or 0.2
% acetic acid
Group
Physiological saline
Control
Equisetum arvense (EA)
Acetic acid solution (0.2%)
Control
Equisetum arvense (EA)
a
b
c
p < 0.01; p < 0.01; p < 0.05
Interval (min)
a
(mean ± SD)
Maximum
Bladder
Contraction
Pressure (cm
H2O)
b
(mean ± SD)
Baseline intravesical
pressure (cm H2O)
c
(Mean ± SD)
Duration (min)
(mean ± SD)
6.2±0.9
8.1±1.2
44.2±5.4
51.3±8.8
6.4±1.3
7.9±1.6
0.9±0.3
0.9±0.3
3.9±1.2
7.3±1.7
48.6±1.7
49.3±2.1
5.9±1.6
8.1±2.1
0.9±0.4
1.3±0.3
Table-2: Effect of Equisetum arvense ethanol root extract on blood pressure and bladder weight
Parameter
Blood pressure
(mm Hg)
Systolic
Mean
Diastolic
Bladder weight (g)
Control
(mean ± SD)
101 ± 4
94 ± 4
81 ± 3
0.16 ± 0.03
Urinary ATP measurement
Before acetic acid solution infusion into the
bladder, the measured urinary ATP revealed no
differences between the control group and EA
group. However, after the 0.2 % acetic acid
solution infusion into the bladder, the urinary
ATP-to-CRE (CRE = creatinine) ratio was
considerably higher on days 0 to 3 with a 5,556
Equisetum arvense
(mean ± SD)
109 ± 5
97 ± 3
86 ± 3
0.15 ± 0.01
%, 429 % and 150 % increase in the control
group as compared with the level before acetic
acid infusion (p = 0.01, p < 0.001 and p < 0.05
respectively, Fig 1 and Table 3). In the EA group,
the urinary ATP-to-CRE ratio was also
considerably higher on days 0 and 1 with a 3,432
% and 150 % increase compared with that before
acetic acid solution infusion (p < 0.01 and p <
0.012 respectively, Fig. 1 and Table 3). However,
Trop J Pharm Res, August 2015; 14(8): 1454
Zhang et al
on comparing the control group with the EA
group after acetic acid infusion, it appeared that
the urinary ATP-to-CRE ratio was significantly
higher on days 0 - 3 in the control group as
compared to the EA group. However, after 6
days the urinary ATP-to-CRE ratio shifted to
baseline and no difference was observed
between the two groups (control and EA group).
Major compounds of extract
The phytochemical analysis of the E. arvense
root ethanol extract was carried out by LC–ESIMS as well as HPLC-DAD techniques. The
extract was run under both positive and negative
ESI-MS conditions and it showed several major
and minor ionic species. The seven chemical
constituents identified were apigenin (1), luteolin
(2),
luteolin-5-O-β-D-glucopyranoside
(3),
isoquercetin (4), apigenin-5-O-glucoside (5),
ursolic acid (6), and oleanolic acid (7) (Fig 2).
These phytochemicals have already been
reported previously in this medicinal plant [9-12].
The total ion MS chromatogram (TIC) and HPLC
3-D plot are shown in Figs 3 and 4, respectively.
The minor peaks in the chromatogram
corresponded to some impurity with unusual
mass fragmentation pattern, hence were not
identified. Fragmentation of the major peaks was
used for the identification of compounds. The
identification of the chemical compounds was
also carried out by comparing the molecular ion
peaks along with the MS fragmentation pattern
with those of the literature.
Fig 2: Phytochemical constituents identified in the ethanol root extract of Equisetum arvense
Table 3: Effect of Equisetum arvense ethanol root extract on the biochemical profile of rat
Parameter
Serotonin (pg/ml)
Control
(mean ± SD)
2,186 ± 350
Equisetum extract
(mean ± SD)
1,877 ± 544
Dopamine (pg/ml)
297 ± 46
221 ± 54
Adrenaline (pg/ml)
19,006 ± 5500
12,051 ± 3561
Noradrenaline (pg/ml)
2,305 ± 643
1,231 ± 321
a
b
Trop J Pharm Res, August 2015; 14(8): 1455
Zhang et al
Table 4: Effect of ethanol extract of Equisetum arvense extract on urinary ATP level after 0.2 % acetic acid
stimulation
Variable
Mean ± SD ATP
(mol/mg CRE x E-10)
Control
Equisetum extract
Before 0.2% acetic acid
stimulation
After 0.2% acetic acid
stimulation
Day 0
12.2 ± 7.6
6.7 ± 5.4
726.9 ± 432.12
321.6 ± 211.2
<0.05
Day 1
87.1 ± 32.5
44.5 ± 21.4
>0.01
Day 2
35.28 ± 11.6
14.31 ± 4.6
>0.01
Day 3
32.1 ± 11.7
7.5 ± 2.7
>0.05
Day 7
6.5 ± 3.2
2.1 ± 1.3
>0.05
P value
ATP=Adenosine triphosphate, CRE= Creatinine, ATP-to-CRE (CRE = creatinine)
Fig 3: LC-MS chromatogram of the ethanol extract of Equisetum arvense indicating the different peaks for the
pytochemical constituents
Fig 4: HPLC 3-D plot of the bioactive extract of Equisetum arvense
Trop J Pharm Res, August 2015; 14(8): 1456
Zhang et al
DISCUSSION
Many disorders today are being treated using
complementary and alternative medicine, and
consumption of herbal ingredients is on a rise
globally, mainly due to the viewpoint that herbal
medicines have less side-effects compared to
orthodox medicines. Serenoa repens is one such
herbal medicinal agent used against various
urological disorders. Published literature reveals
that men with BPH exhibited a significant
improvement in the urinary peak flow rate and
decreased nocturia after the administration of
Serenoa repens, which is a lipid sterol extract
from dwarf palms [13].
In a one-year international study, comparing
Serenoa repens with an α-1-adrenergic receptor
antagonist for BPH, it was observed that both
have an equivalent effect in men with BPH and
LUTS for one year [14]. Another traditional
Kampo drug called Gosha-jinki-gan, which
comprises of 10 medicinal herbs, is also
considered very effective for various urological
disorders like LUTS [15,16].
In one study, Gosha-jinki-gan was reported to
increase
the
interval
between
bladder
contractions and enhance capacity of the
bladder. This drug is thought to have
anticholinergic activity, which is capable of
blocking bladder contraction induced by
cholinergic nerve stimulation. Eviprostat, another
herbal drug which is a cocktail of many medicinal
plant extracts, has the effect of decreasing
urethral pressure and increasing voided volume.
This cocktail has also been reported to inhibit
edema in the prostate gland and inflammation of
the bladder mucosa in BPH patients [17]. The
medicinal plants comprising eviprostat were
initially used as folk medicine for many urological
symptoms like BPH, cystitis, epididymitis,
prostatitis etc.
In this study, we have tried to delineate the
effects of E. arvense and its influence rat bladder
activity by carrying out a set of examinations,
comprising
of
continuous
cystometry,
biochemical tests, urinary ATP and blood
pressure measurement. No such previous
reports are documented in the literature on this
medicinal plant. We evaluated the changes in the
body weight caused by the administration of EA
diet. It was observed that on EA administration,
plasma catecholamine concentration was
lowered. Furthermore, plasma adrenaline and
noradrenaline concentrations were considerably
lower in the EA group as compared to the control
group. During continuous cystometry with
physiological
saline,
maximum
bladder
contraction pressure was significantly lower in
the control group as compared to EA group. This
may be due to the effect of Equisetum arvense
on the bladder, bladder neck and urethra. The
effect of the decrease in adrenaline,
noradrenaline and urinary ATP levels is reflected
in the difference between maximum bladder
contraction pressure of control (44.2) and EA
group (51.3).
Further, in order to create a bladder inflammation
model, 0.2 % acetic acid solution was infused
into the bladder. With acetic acid solution
continuous cystometry, the time interval between
bladder contractions became significantly shorter
and maximum bladder contraction pressure was
much higher in the control group in comparison
to physiological saline group. However, no
considerable differences in the cystometric
parameters between the infusion of physiological
saline and 0.2 % acetic acid were observed in
EA group. In this study, we also observed that
EA treatment inhibits an increase in the urinary
ATP-to-CRE ratio after bladder stimulation. It is
also well known that acetylcholine and ATP are
produced by bladder epithelial cells (17). It has
also been reported recently that ATP secreted by
bladder epithelial cells might influence bladder
activity. Therefore, the inhibitory effect of EA on
bladder activity may be linked to blocking ATP
release from the bladder epithelial cells.
Furthermore, the level of bladder activation may
be recognized by using increased urinary ATP
release as a marker. Equisetum arvense has
been reported to act as a potent radical
scavenger and these radical scavenging
capabilities may contribute to their antiinflammatory action and blocking of ATP release.
Apart from the effects of EA on bladder activity,
photochemical analysis of EA by ESI-MS showed
several major and minor ionic species. The
seven chemical constituents identified were
apigenin (1), luteolin (2), luteolin-5-O-β-Dglucopyranoside (3), isoquercetin (4), apigenin-5O-glucoside (5), ursolic acid (6), and oleanolic
acid. These will need to be evaluated for their
biological effects in future studies.
CONCLUSION
The findings of this study suggest that Equisetum
arvense ethanol root extract influences the
urinary bladder activity by decreasing adenosine
triphosphate release and thus can potentially be
used as a therapeutic agent against bladder
disorder. It may find application in the therapeutic
efforts to reduce overactivty of urinary bladder
which has only a limited number of therapies
available at present.
Trop J Pharm Res, August 2015; 14(8): 1457
Zhang et al
ACKNOWLEDGEMENT
of stems from Equisetum arvense L. in mice.
Pharmacol Res 2004; 49(3): 239-243.
The authors would like to thank Shandong
Province Natural Science Foundation of China
(project no. ZR2009CL027), Science and
Technology Star Plans Foundation of Jinan
Technology Bureau of China (project no.
20100118) as well as Medical Foundation of
Shandong Academy of Medical Science (project
no. 201023) for providing financial support for
this research work.
9. Suzuki K, Homma T. Isolation and chemical structure of
flavonoids from horsetail (Equisetum arvense L). J
Adv Sci 1997; 9: 104-105.
10. Pietta P, Mauri P, Bruno A, Rava A, et al. Identification of
flavonoids from Ginkgo biloba L., Anthemisnobilis L.
and Equisetum arvense L. by high-performance liquid
chromatography with diode-array UV detection. J
Chromatogr 1991; 553: 223-31.
11. Ganeva Y, Chanev C, Dentchev T. Triterpenoids and
sterols
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